The cytokine TNF, the pathogen recognition Toll-like receptors (TLRs) and the nucleotide binding oligomerization domain (NOD) proteins mediate host defense against infection in part by activating the transcription factor NF-B. Our published work reveals Rip1 as a critical mediator of the TNF- and TLR3/4, Trif-dependent NF-B pathways and the ubiquitin modification of Rip1 is essential for TNF- induced NF-B activation and cytokine production. We find Rip1-deficient cells impaired in their type I interferon response to viral infection, revealing that Rip1 contributes to Rig-I/Mda5 anti-viral signaling. Our recent studies reveal a novel regulatory role for Rip1 in the ubiquitination and activation of the interferon regulatory factor 7 (IRF-7), a transcription factor critical for type I interferon production. Therefore, we hypothesize that Rip1 and potentially polyubiquitinated Rip1 mediate anti-viral innate immune responses by regulating NF-B and IRF-7 activity. To support this hypothesis, we will test whether a Rip1-deficiency impairs innate anti-viral responses in vivo and will infect Rip1-deficient macrophages and dendritic cells with multiple classes of viruses to determine how Rip1 regulates the transcriptional activity of IRF-7 (Aim 1). Activation of the TNF, Trif or Rig-I/Mda5 pathways stimulates Rip1 polyubiquitination, hence we will test whether polyubiquitinated Rip1 is required for the activation of NF-B and/or IRF-7 in virally infected cells and will determine whether an inability to ubiquitin modify Rip1 results in impaired innate anti-viral responses (Aim 2). Similarly, our studies on the related Rip1 protein Rip2 find endogenous Rip2 polyubiquitinated in MDP-stimulated macrophages, suggesting that the Nod2 pathway is ubiquitin-regulated. We hypothesize that polyubiquitinated Rip2 is required for Nod2-mediated NF-B activation and for innate anti-bacterial immune responses and predict that ubiquitin deregulation contributes to NOD2-associated human inflammatory diseases. To test this hypothesis, we will identify the critical ubiquitin site on Rip2 and will test whether polyubiquitinated Rip2 is required for Nod-mediated NF-B activation and will determine how expression of NOD2 alleles associated with human inflammatory diseases affect the recruitment and polyubiquitination of Rip2 (Aim3). Collectively, our studies suggest that innate immune responses are ubiquitin regulated, raising the possibility that the enzymes responsible for the ubiquitin modification of Rip proteins may be targeted therapeutically to treat infectious disease or chronic inflammatory disease.